Healthy sleep/wake cycles depend on the proper integration of neural circuits that control arousal and sleep. Insight on the interactions between these circuits will promote our ability to treat sleep disorders. Recent published work shows that a subset of weakly circadian neurons in the Drosophila pacemaker circuit is physiologically responsive to sensory input and contributes to behavioral arousal (Sheeba et al., 2008; Parisky et al., 2008; Shang et al., 2008). An integrative combination of molecular, physiological, and behavioral approaches to illuminate an interface between circadian and arousal neural circuits is proposed. Towards this goal, novel methods to electrophysiologically record Drosophila pacemaker neurons in whole brain preparations and in dissociated neuronal cultures are described. Another key recent technical advance is to image per promoter cycling for the entire PER-expressing central brain circadian circuit at single cell resolution for multiple days. This was achieved by combining a highly sensitive low-light imaging system with a robust long-term Drosophila whole brain culture system that we recently co-developed (Ayaz et al., 2008). To the best of our knowledge, this is the first example of a preparation that permits long-term multi-day imaging of a sensory-enabled entire neural circuit. The Specific Aims are: Specific Aim 1. Determine the specificity, coupling, and underlying signal transduction of CRYPTOCHROME-dependent light-induced rapid changes in pacemaker neuron firing rate. Specific Aim 2. Determine whether large lateral ventral neurons contribute to behavioral arousal. Specific Aim 3. Determine the pattern of PER cycling for entire circadian circuit in response to light activation and exogenously induced firing of the large LNv arousal neurons at high spatial and temporal resolution by whole brain per-luc imaging. These studies will reveal critical operations that occur between arousal and circadian circuits. The proposed work will likely provide new insights for mammalian circadian biology in a rapid and cost effective manner and the molecular genetic and physiological tools described herein will be used in the future by other laboratories for studying neural circuits and disorders of electrical excitability.